This invention relates to the field of cell biology, more specifically the teichoic acid pathway. Genes and proteins related to this pathway include: Teichoic Acid Polymerase (or TAP), and CDP-Glycerol:Poly(glycerophosphate) Glycerophosphotransferase.
A. L. Honeyman, G. C. Stewart, xe2x80x9cIdentification of the protein encoded by rodC, a cell division gene from Bacillus subtilisxe2x80x9d Mol. Microbiol. (1988) 2:735-741.
A. L. Honeyman, G. C. Stewart, xe2x80x9cThe nucleotide sequence of the rodC operon of Bacillus subtilis. Mol. Microbiol. (1989) 3:1257-1268.
C. Mauel, M. Young, P. Margot, D. Karamata xe2x80x9cThe essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesisxe2x80x9d Mol. Gen. Genet. (1991) 215:388-394.
C. Mauel, M. Young, D. Karamata, xe2x80x9cGenes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription unitsxe2x80x9d J. Gen. Microbiol. (1991) 137:929-941.
Y. S. Park, T. D. Sweitzer, J. E. Kison, C. Kent. xe2x80x9cExpression, purification, and characterization of CTP:Glycerol-3-phosphate cytidyltransferase from Bacillus subtilis.xe2x80x9d J. Biol. Chem. (1993) 268:16648-16654.
The spread of antibiotic resistance in gram positive pathogenic bacteria is a serious problem which is only beginning to be registered in the clinic. The incidence of drug resistance is increasingxe2x80x94especially in Staphylococcus aureus, Streptococcus pneumonia, and the enterococci. Methicillin resistant S. aureus (MRSA), penicillin resistant S. pneumoniae, and vancomycin resistant enterococci, pose a serious threat to compromised patients. Vancomycin is the only antibiotic effective against MRSA. See, C. T. Walsh, xe2x80x9cVancomycin resistance: decoding the molecular logicxe2x80x9d Science (1993) 261:308-309; I. R. Friedland, xe2x80x9cTherapy of penicillin- and cephalosporin-resistant pneumococcal infectionsxe2x80x9d Trends Clinic Pract. (1993) 25:451-455 and S. Dutka-Malen, and P. Courvalin, xe2x80x9cUpdate on glycopeptide resistance in enterococcixe2x80x9d Antimicrob News (1990) 7:81-88.
The cell wall teichoic acid pathway is found in the majority of gram positive bacteria, and studies with Bacillus subtilis have revealed that it is essential to cell viability. See, C. Mauel, M. Young, P. Margot, D. Karamata, xe2x80x9cThe essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesisxe2x80x9d Mol Gen Genet (1991) 215:388-394. The essential nature of cell wall teichoic acid may be due to the covalent attachment that it forms with peptidoglycan.
Cell wall teichoic acid, like peptidoglycan, is synthesized at the outer surface of the cell membrane using a nucleotide precursor (CDPglycerol) as the building block. Teichoic acid is a polymer of polyglycerolphosphate that is covalently attached to the peptidoglycan of gram positive bacteria. The enzyme CDP-Glycerol: Poly(glycerophosphate) glycerophosphotransferase catalyzes the polymerization of glycerolphosphate monomers from CDP-glycerol into a chain of polyglycerolphosphate linked via 1,3-phosphodiester bonds. Lipoteichoic acid is a related polymer of polyglycerolphosphate which is anchored to the cell membrane but is not attached to peptidoglycan.
There is an obvious clinical need for new antimicrobial agents which inhibit novel targets. In order to screen for unique inhibitors, essential metabolic pathways of gram positive pathogens, such as the cell wall teichoic acid pathway must be identified and their respective enzymes studied, cloned and made into useful assays and screens in order to identify novel antimicrobial agents.
This invention discloses a method of measuring and assaying the activity of the TAP enzyme. This invention also demonstrates how a common commercially available material may be used as a substrate for an important biological reaction that has previously had no substrate available for evaluating this reaction. This invention teaches the researcher and clinician that lipoteichoic can be used as a substrate to elucidate the presence and even the activity of the TAP enzyme. An embodiment of this invention is the application of this teaching to create an assay that enables one to monitor the activity of the TAP enzyme.
This invention also discloses, for the first time, the sequence of an active TAP enzyme and the nucleic acid sequence of the DNA that codes for this sequence.
This invention includes: the entire DNA sequence shown in FIG. 3 and Sequence Listing I.D. no. 1, and the DNA from residues 4 to 2274, first to last restriction site, and the DNA residues 24 to 2264. The coding DNA sequence shown in FIG. 3, alternatively named, xe2x80x9cthe rodC gene.xe2x80x9d The DNA sequences corresponding to the sequence in FIG. 3 where the residue at position 1872 is thymine in place of cytosine.
A bacterial DNA sequence that is capable of hybridizing to the DNA sequence of FIG. 3, under standard stringent conditions, to about 70 or more including, 75, 80, 85, 90, 95 or greater percent homology and having the ability to catalyze the reaction of CDP-glycerol plus H2O into teichoic or lipoteichoic acid.
The DNA sequence from Staphylococcus aureus that codes for the protein or protein sequence fragment from Staphylococcus aureus having at least 70% homology to related fragments described by FIG. 3 and FIG. 4, and that yield fragments of 7.0 kb, 5 kb, and 4.2 kb after EcoRI digest, or that yield fragments of 4.5, 3.3, 2.8 kb, after HindIII digest.
In addition to the DNA sequence, this invention describes various mutants, including: A collection of randomly mutated rodC genes. A selection of one or more randomly mutated rodC genes. A collection of bacteria having randomly mutated rodC genes. A selection of one or more bacteria having a random mutation selected from the collection of bacteria. The mutated bacteria selected from a mutant form of B. subtilis or S. aureus. 
Various proteins and peptide fragments from the expressed DNA are also described. The entire protein sequence shown in FIG. 3 and FIG. 4, Sequence I.D. NO. 2, the protein sequence from residues 1-746, and the protein sequence shown in FIG. 3 and FIG. 4 where valine is the amino acid at position 616 in place of alanine. Also described are the protein sequence fragment from Staphylococcus aureus having at least 70% homology to related fragments described by FIG. 3 and FIG. 4, that yield fragments of 7.0 kb, 5 kb, and 4.2 kb after EcoRI digest; and the protein sequence fragment from Staphylococcus aureus having at least 70 % homology to related fragments described by FIG. 3 and FIG. 4 that yield fragments of 4.5, 3.3, 2.8 kb, after HindIII digest. The protein disclosed in the Southern Blot shown in FIG. 2 is described as well.
In addition to the DNA and proteins disclosed herein, this invention comprises various intermediates, intermediate vectors, plasmids and transformed or mutated cell lines. This invention comprises the DNA of the sequence disclosed in FIG. 3 incorporated into a vector selected from a cloning vector, a shuttle vector or an expression vector, any of these vectors may be plasmid vectors. The cloning vector or plasmid can be selected from any widely available or commercially available plasmids. The plasmid can be any suitable pUC type or pBR type of plasmid, such as pUC18, or pUC19, or any other suitable plasmid such as pBR322. The vector may be a typical shuttle, vector The shuttle vector may be a plasmid such as, pMK4, or pYL112xcex94119. An expression vector may also he used, the expression vector is a plasmid with a very strong promoter, such as the following very strong promoters: pTrc99A, pDR540, or pET-21(+). In this nomenclature pTRC99A would be the name of the plasmid. Each plasmid used for expression of proteins has a unique promoter as follows: pTRC99A (trc promoter), pDR540 (tac promoter), pET-21(+) (T7 promoter).
Examples of plasmids would be a plasmid named pRODCAP18 comprising the cloned rodC gene, placed into the cloning vector, pUC18, the plasmid named pMKRODC comprising the the cloned rodC gene, placed into the shuttle vector pMK4, the plasmid where the plasmid was created from a rodC gene excised from a pRODCAP18 plasmid, the plasmid selected from the plasmids named pBSRODC1 or pBSRODC1, comprising the rodC gene, placed into an expression vector with a strong promoter that is pTrc99A and plasmids created where the rodC gene is excised from the pMKRODC plasmid.
These plasmids may be used to create transformed bacterial cells and collections of mutant cells and plasmids may be easily created. So there are further descriptions of a bacterial cell transformed with the various disclosed plasmids and a bacterial cell that is an E. coli cell, and an E. coli cell variously transformed that is of type DH10B.
New and novel assays are also disclosed and a most important assay disclosed herein does NOT demand the newly discovered DNA and protein although in some embodiments they are required. This invention comprises: A method of measuring the activity of the TAP enzyme comprising combining CDP-glycerol plus H2O or water plus TAP enzyme plus lipoteichoic acid and measuring the amount of lipoteichoic acid formed. In one embodiment the CDP-glycerol is radioactive CDP-glycerol, in one embodiment the activity of the TAP enzyme comprises combining radioactive CDP-glycerol plus H2O plus TAP enzyme plus lipoteichoic acid plus streptavidin SPA beads and a suitable lectin such as a wheat germ agglutinin and measuring the amount of radioactive lipoteichoic acid formed as indicated by measuring the lectin bound to the SPA bead. In all these embodiments the radioactive CDP-glycerol cna be made from [3H]glycerol-3-phosphate (a.k.a. [3H]glycerophosphate). A preferred method of practicing any of these assays is to treat the lipoteichoic acid to remove the alanine residues before using it in the assays, that is, before combining with the other ingredients, the lipoteichoic acid is treated to remove alanine. These assays may be used to measure the activity of TAP enzyme when it is from an impure preparation or the methods may be used where the TAP enzyme is the enzyme disclosed in FIG. 3 and FIG. 4, or Sequence ID listing number 2. The assays herein may be configured into kits for ease of application.
Also disclosed is a method of using lipoteichoic acid as a substrate for the enzymatic reaction catalyzed by the TAP protein. Lipoteichoic acid, unlike teichoic acid, is commercially available and thus makes an excellent substrate. The lipoteichoic acid can serve as an acceptor of CDP[3H]glycerol. The TAP protein can be obtained from crude sources or extracts, preferably the lipoteichoic acid is prepared from B. subtilis, S. aureus, or E. faecalis, or it can be the TAP protein described in FIG. 3 and FIG. 4 and Sequence Listing I.D. no. 2, or a protein having at least about 70% homology to that protein.
A diagnostic kit utilizing the TAP enzyme and CDPglycerol to detect and monitor disease caused by gram positive bacteria can be created using the information disclosed herein. Following appropriate instructions from such a kit, a portion of the biological sample which is thought to contain lipoteichoic acid could be added to TAP and CDPglycerol, incubated for an hour or so, and the transfer of glycerol-3-phosphate from CDPglycerol to lipoteichoic acid present in the sample could be detected using the precipiation assay described below under xe2x80x9cPrecipitation Assay.xe2x80x9d